The United States is in the midst of an opioid epidemic and risk is especially high in veterans. Current therapies consist mainly of alternative opioids, but their efficacy is limited. Thus, new treatments for pain and addiction are in high demand. Improved knowledge of the mechanisms underlying opioid addiction and relapse could help predict who might be a risk for addiction, and help to develop better therapies for those already battling addiction and relapse. The reinforcing effects of opioids depend in large part on the nucleus accumbens (NAc) and mu opioid receptors, which are expressed at glutamatergic synapses on medium spiny neurons in the NAc. Opioids and other drugs of abuse can hijack these synapses and alter their number, morphology, and glutamate receptor subunit composition. These changes are thought to produce abnormal synaptic states that underlie addiction, withdrawal, craving, and relapse. We recently identified a novel signaling mechanism that can influence the synaptic and behavioral effects of opiate drugs. This mechanism involves protons released from presynaptic neurotransmitter-containing vesicles, activation of the post-synaptic acid sensing ion channel, ASIC1A, and pH buffering at the synapse by carbonic anhydrase 4 (CA4). Our previous studies and pilot data suggest that ASIC1A plays a critical role in stabilizing glutamatergic synapses in the NAc, and that loss of ASIC1A increases vulnerability to synaptic abnormalities induced by opioids, as well as by cocaine. Interestingly, reducing pH buffering by disrupting CA4, increases the inward synaptic Na+ currents mediated by ASIC1A and appears to protect against the synaptic rearrangements thought to contribute to addiction, craving, and relapse. Here we propose to extensively test the degree to which CA4 and ASIC1A regulate opioid-induced synaptic abnormalities in the NAc, and influence opioid-reinforced behaviors. Specific Aim 1 proposes to test the effects of opioids on synaptic physiology in mice lacking ASIC1A, CA4, or both. Specific Aim 2 proposes to test effects of opioids in these mice in multiple behavioral paradigms including opioid self-administration, withdrawal, and relapse-related behaviors. We hypothesize that disrupting ASIC1A will alter synaptic and behavioral effects of opioids, disrupting CA4 will protect against these effects, and that effects of CA4 disruption will depend on ASIC1A. Together the experiments in this proposal will pave the way to a better understanding of the neurobiology underlying opioid addiction. Moreover, the knowledge gained from these studies could suggest new ways to treat opioid addiction through non-opioidergic pathways, for example by manipulating brain pH, ASICs, or carbonic anhydrase, for which several inhibitors are already approved for human use.